Probe rank approaches for gene selection in oligonucleotide arrays with a small number of replicates

MOTIVATION: One major area of interest in analyzing oligonucleotide gene array data is identifying differentially expressed genes. A challenge to biostatisticians is to develop an approach to summarizing probe-level information that adequately reflects the true expression level while accounting for probe variation, chip variation and interaction effects. Various statistical tools, such as MAS and RMA, have been developed to address this issue. In these approaches, the probe level expression data are summarized into gene level data, which are then used for downstream statistical analysis. Since probe variation is often larger than chip variation and there is also a potential interaction effect between probe affinity and treatment effect, strategies such as a gene level analysis, may not be optimal. In this study, we propose a procedure to analyze probe level data for selecting differentially expressed genes under two treatment conditions (groups) with a small number of replicates. The probe level discrepancy between two groups can be measured by a difference of the percentiles of probe perfect-match (PM) ranks or of probe PM weighted ranks. The difference is then compared with a pre-specified threshold to determine differentially expressed genes. The probe level approach takes into account non-homogenous treatment effects and reduces possible cross-hybridization effects across a set of probes. RESULTS: The proposed approach is compared with MAS and RMA using two benchmark gene array datasets. Positive predictivity and sensitivity are used for evaluation. Results show the proposed approach has higher positive predictivity and higher sensitivity. AVAILABILITY: Available on request from the authors. CONTACT: dtchen@uab.edu.     

Change in the Cortical Complexity of Spinocerebellar Ataxia Type 3 Appears Earlier than Clinical Symptoms

Patients with spinocerebellar ataxia type 3 (SCA3) have exhibited cerebral cortical involvement and various mental deficits in previous studies. Clinically, conventional measurements, such as the Mini-Mental State Examination (MMSE) and electroencephalography (EEG), are insensitive to cerebral cortical involvement and mental deficits associated with SCA3, particularly at the early stage of the disease. We applied a three-dimensional fractal dimension (3D-FD) method, which can be used to quantify the shape complexity of cortical folding, in assessing cortical degeneration. We evaluated 48 genetically confirmed SCA3 patients by employing clinical scales and magnetic resonance imaging and using 50 healthy participants as a control group. According to the Scale for the Assessment and Rating of Ataxia (SARA), the SCA3 patients were diagnosed with cortical dysfunction in the cerebellar cortex; however, no significant difference in the cerebral cortex was observed according to the patients’ MMSE ratings. Using the 3D-FD method, we determined that cortical involvement was more extensive than involvement of traditional olivopontocerebellar regions and the corticocerebellar system. Moreover, the significant correlation between decreased 3D-FD values and disease duration may indicate atrophy of the cerebellar cortex and cerebral cortex in SCA3 patients. The change of the cerebral complexity in the SCA3 patients can be detected throughout the disease duration, especially it becomes substantial at the late stage of the disease. Furthermore, we determined that atrophy of the cerebral cortex may occur earlier than changes in MMSE scores and EEG signals.     

Isolation and partial characterization of siderophore mutants ofAzotobacter vinelandii

Azotobacter vinelandii was mutagenized with ethyl methanesulfonate, and colonies that did not produce the fluorescent yellow-green pigment that is characteristic of the wild type were selected. All 32 stable nonfluorescent mutants failed to secrete the siderophore azotobactin and were also impaired to some extent in the production of the second majorA. vinelandii siderophore, azotochelin. Mutants also showed differences in their capacity to grow on medium supplemented with either 200 M bipyridyl or 200 M Fe (III). In the absence of iron, an 84-kilodalton outer membrane protein, which is a major derepressed component, was missing in some of the mutants. Thus, siderophore production inA. vinelandii appears to be a highly integrated system in which the syntheses of azotobactin and azotochelin are functionally coupled.     

Genetic diversity of soil microorganisms assessed by analysis ofhsp70 (dnaK) sequences

The genetic diversity of a soil microbial community was assessed by analysis of clonedhsp70 sequences. A clone library was generated by polymerase chain reaction-mediated amplification of a 650-base pair fragment of thehsp70 gene, using DNA extracted from soil, without culturing the microorganisms. Fifty-five random clones were sequenced and their amino acid sequences deduced. Analysis of the amino acid sequence of the clones revealed the presence of signature sequences in common with known prokaryotic and lower eukaryotic HSP70 homologs. None of the 55 analyzed sequences were identical to each other or to a published sequence. These results confirm the presence of considerable genetic diversity within soil microbial communities, the major proportion of which remains uncharacterized.     

The striking effects of spacer and terminator sequences on the synthesis of porcine growth hormone inEscherichia coli

Summary Two types of expression vectors containing theP _tac orP _L promoters, synthetic ribosomalbinding sequences (RBS), and a transciptional terminator (rrnBT₁T₂) were constructed for the production of porcine growth hormone (pGH) inEscherichia coli. The results demonstrated that the nucleotide sequence between the RBS and the start codon, ATG, was critical for the yields of pGH. The rrnBT₁T₂ terminator was essential for the efficient expression ofpGH in theP _tac -driven vectors, but reduced the synthesis of pGH in theP _L -vectors. A leaky repression was observed in the JM103 cells containing theP _tac -plasmids.     

PEG molecular weight and lateral diffusion of PEG-ylated lipids in magnetically aligned bicelles

Lateral diffusion coefficients of PEG-ylated lipids with three different molecular weight PEG groups (1000, 2000 and 5000) were measured in magnetically-aligned bicelles using the stimulated echo (STE) pulsed field gradient (PEG) (1)H nuclear magnetic resonance (NMR) method. At concentrations below the PEG "mushroom-to-brush" transition, all three PEG-ylated lipids exhibited unrestricted lateral diffusion, with lateral diffusion coefficients comparable to those of corresponding non-PEG-ylated lipids and independent of PEG molecular weight. At concentrations above this transition, lateral diffusion slowed progressively with increasing concentration of PEG-ylated lipid as a result of surface crowding. As well, the lateral diffusion coefficients exhibited a pronounced decrease with increasing PEG group molecular weight and a diffusion-time dependence indicative of obstructed diffusion. We conclude that, while lateral diffusion of PEG-ylated lipids within lipid bilayers is determined primarily by the hydrophobic anchoring group, when crowding at the lipid bilayer surface becomes significant, the size of the extra-membranous domain, in this case the PEG group, can influence lateral diffusion, leading to decreased diffusivity with increasing size and producing obstructed diffusion at high crowding. These findings imply that similar considerations will pertain to lateral diffusion of membrane proteins with large extra-membranous domains.     

Exposure of phosphatidylserine on Leishmania amazonensis isolates is associated with diffuse cutaneous leishmaniasis and parasite infectivity

Diffuse cutaneous leishmaniasis (DCL) is a rare clinical manifestation of leishmaniasis, characterized by an inefficient parasite-specific cellular response and heavily parasitized macrophages. In Brazil, Leishmania (Leishmania) amazonensis is the main species involved in DCL cases. In the experimental model, recognition of phosphatidylserine (PS) molecules exposed on the surface of amastigotes forms of L. amazonensis inhibits the inflammatory response of infected macrophages as a strategy to evade the host immune surveillance. In this study, we examined whether PS exposure on L. amazonensis isolates from DCL patients operated as a parasite pathogenic factor and as a putative suppression mechanism of immune response during the infection. Peritoneal macrophages from F1 mice (BALB/c×C57BL/6) were infected with different L. amazonensis isolates from patients with localized cutaneous leishmaniasis (LCL) or DCL. DCL isolates showed higher PS exposure than their counterparts from LCL patients. In addition, PS exposure was positively correlated with clinical parameters of the human infection (number of lesions and time of disease) and with characteristics of the experimental infection (macrophage infection and anti-inflammatory cytokine induction). Furthermore, parasites isolated from DCL patients displayed an increased area in parasitophorous vacuoles (PV) when compared to those isolated from LCL patients. Thus, this study shows for the first time that a parasite factor (exposed PS) might be associated with parasite survival/persistence in macrophages and lesion exacerbation during the course of DCL, providing new insights regarding pathogenic mechanism in this rare chronic disease.     

Synthesis and self-assembly of a DNA-conjugated poly(propylene glycol) derivative

Due to several characteristic features of DNA (e.g., genetic coding transfer and nanoscaled accuracy or so), DNA based compounds have been recently highlighted in a variety of research fields, including physics, chemistry, engineering or so. To date, they have been expanded into a hybrid form conjugated with conventional polymer groups. Such synthetic hybrid conjugates can be organized into multi-dimensional nanoor micro-structures; rod, sheet, and spherical shaped nanomaterials [1]. In this study, we showed a novel block copolymer composed of DNA and poly(propylene glycol) (PPG) (designated as DNA-b-PPG) for the self-assembled construction into three-dimensional vesicular structures.     

Leishmania donovani: expression and characterization of Escherichia coli-expressed recombinant chitinase LdCHT1

Leishmania parasites produce chitinase activity (EC. 3.2.1.14) thought to be important in parasite-sandfly interactions and transmission of the parasite to the vertebrate host. Previous observations have suggested that parasite chitinases are involved in degradation of the sandfly peritrophic matrix and the chitinous layer of the cardiac valve cuticle. This chitinase activity is thought to produce an incompetent pharyngeal valve sphincter and a route of egress that allow Leishmania promastigotes to be regurgitated into the site of blood feeding. In the studies reported here, enzymatically active L. donovani chitinase LdCHT1 was expressed as a thioredoxin fusion protein in Escherichia coli strain AD494 (DE3). Recombinant LdCHT1 had a predominantly endochitinase activity, in contrast to previous reports of both exo- and endochitinase activities in axenic culture supernatants of diverse Leishmania spp. promastigotes. The predominant endochitinase activity of recombinant LdCHT1 is consistent with the presumed function of the enzyme in disrupting chitinous structures in the sandfly digestive system to allow transmission.     

Binding of calmodulin to the microfilament network correlates with induction of a macrophage tumoricidal response

Induction of mouse peritoneal macrophage cytotoxicity against SV3T3, a line of virally transformed mouse cells correlated with the distribution of cytoplasmic calmodulin in the macrophages. The organization of the cytoskeleton was examined by fluorescent microscopy and by transmission electron microscopy, using immunogold tagging after Triton-X-100 (TX-100) extraction of the macrophages. Macrophages that had been activated to a tumoricidal state in vivo by vaccinia virus or in vitro by lymphokine stimulation displayed cytoskeletal networks that were more extended and weblike than did resident macrophages. The organization of microfilaments and microtubules in the cytoskeleton was displayed by using either anti-actin or anti-tubulin. Immunogold labeling of tumoricidal macrophage cytoskeletons with anti-calmodulin revealed strong binding to the microfilament network and no binding to microtubules. Anti-calmodulin reacted weakly with the cytoskeletal network of resident macrophages, and this was not demonstrably greater than the reaction with normal sheep serum. However, resident macrophages displayed a high density of calmodulin (CAM) associated with unidentifiable structures in the perinuclear region when reacted with anti-calmodulin. These characteristic distributions of CAM in resident and activated macrophages was confirmed by immunofluorescence. The total and cytoskeletal-associated amounts of calmodulin per unit of protein were determined by radioimmune assay and 125I labeling followed by SDS-PAGE. No statistically significant differences were detected between resident and activated macrophages in either the total cell or cytoskeleton fractions. In summary, our results suggest that induction of tumoricidal activity of mouse peritoneal macrophages correlates with the translocation of calmodulin to the microfilament network of the cytoskeleton.